Method of determining and prognosing severity of pregnancy toxemia and method of estimating fetus/placenta function under pregnancy toxemia

ABSTRACT

Provided are: a method for predicting the onset of pregnancy-induced hypertension (PIH) by precisely detecting abnormalities that occur before the onset of PIH (where such abnormalities have been impossible to detect by various conventional testing methods for PIH) while imposing less of a burden on a subject; a method for evaluating a fetus and placental functions in PIH; and a method for detecting PIH, which comprises measuring the level of human lipocalin-type prostaglandin D synthase (L-PGDS) in a body fluid sample collected from a subject.

TECHNICAL FIELD

The present invention relates to a method for determining the severityof and predicting pregnancy-induced hypertension (PIH). Specifically,the present invention relates to a method for conveniently andobjectively determining PIH and a method for predicting the risk of theonset of PIH in a pregnant woman who has not show any clinical symptomsof PIH. Furthermore, the present invention relates to a method forevaluating a fetus and placental functions in PIH.

BACKGROUND ART

Pregnancy-induced hypertension (PIH) is a major cause of death inpregnant and parturient women in addition to bleeding, obstetricalpulmonary embolus, and the like. A method for managing PIH is still animportant issue for obstetricians. However, the cause and morbidity ofPIH remain unknown. Furthermore, the definition and classification ofPIH have not yet been unified worldwide. In Japan, PIH is defined by thepresence of at least one of hypertension, proteinuria, and edema assymptoms during pregnancy, where these symptoms are not simpleaccidental complications of pregnancy. However, hypertension isabsolutely the major symptom that indicates PIH. A case in which edemaalone is exhibited is not said to be PIH. PIH is found in approximately10% of pregnant women (6% to 14%).

PIH is classified into early onset type (onset before pregnancy week 32)and late onset type (onset at and after pregnancy week 32) depending onthe onset timing. Classification of PIH in terms of severity is asfollows. A case in which one or more of hypertension, proteinuria, andedema as symptoms are exhibited, which are all determined to be mild, isdetermined to constitute mild-type PIH. A case in which at least one ofthese symptoms, which is determined to be severe, is exhibited isdetermined to constitute severe-type PIH. In both early onset type andsevere-type PIH, organ damage becomes rapidly worse in both the motherand the fetus, so that prognosis is bad. Thus, these types of PIHrequire strict management.

PIH is diagnosed by examining the symptoms of hypertension, proteinuria,and edema. Specifically, hypertension is determined to exist whensystolic blood pressure is 140 mmHg or more and diastolic blood pressureis 90 mmHg or more. Furthermore, proteinuria is determined to exist when30 mg/dL or more protein is detected in 24 hour urine specimen byEsbach's method or a measurement method according thereto. Edema isdetermined to exist when a tibial crest remains depressed after digitalcompression and body weight gain of 500 g or more is observed during themost recent 1 week of pregnancy. When at least one of these symptoms isobserved, a diagnosis of PIH is made (edited by Shoichi Sakamoto, etal., Pregnancy-induced hypertension, Principles of Obstetrics andGynecology 2: MEDICAL VIEW CO., LTD., 1998: 340-60).

Furthermore, a general PIH case leads to decreased circulating bloodvolume and is associated with hemoconcentration. Thus, plasma proteinconcentration may also be measured. Furthermore, severe PIH often leadsto microthrombus formation that indicates an elevated state of thecoagulation system and the same with respect to the secondaryfibrinolytic system. Thus, factors involved in suchcoagulation-fibrinolytic system, such as blood platelets and D-dimer,are also examined. Furthermore, lipids, hepatic functions, and the likeare also tested if necessary (edited by Shoichi Sakamoto et al.,Pregnancy-induced hypertension. Principles of Obstetrics and Gynecology2: MEDICAL VIEW CO., LTD. 1998: 340-60).

Management of a patient with PIH and therapeutic methods therefore varydepending on severity. However, the severity of PIH is currentlydetermined comprehensively by a combination of the above-describedplurality of testing methods. Therefore, establishment of a method forconveniently and objectively determining severity is desired. Moreover,it is thought that the influence of PIH can be substantially suppressedby strict management when PIH is found at the initial stage. However,detection of PIH at the initial stage is difficult via any of theabove-described testing methods. Currently, no indicator for predictingthe onset of PIH has been established.

Furthermore, when PIH becomes more severe, placental functions willdecrease and the nutritional and oxygen conditions of a fetus will beworse. Determination of such functions is very important indetermination of delivery timing for a fetus. Accordingly, a preciseindicator for evaluating fetuses and placental functions is desired.

Human lipocalin-type prostaglandin D synthase (hereinafter, L-PGDS) isan enzyme that catalyzes isomerization from PGH₂, which is a commonprecursor of various prostaglandins, to PGD₂. L-PGDS is amultifunctional protein because it also has a function of transportingsmall hydrophobic molecules (Urade Y. et al., Prostaglandin D synthase:Structure and Function. Vitam Horm 2000; 58: 89-120). It has beenreported that elevated blood L-PGDS levels(concentrations) are detectedin patients with advanced renal disease (Hoffmann A. et al., MolecularCharacterization of β-trace Protein in Human Serum and Urine: APotential Diagnostic Marker for Renal Diseases. Glycobiology 1997; 7:499-506). Furthermore, the present inventors have revealed that L-PGDSlevels are increased in the body fluids of patients with early renaldisease (before the progression of renal disease) (Hamano K. et al.,Blood Sugar Control Reverses the Increase in Urinary Excretion ofProstaglandin D Synthase in Diabetic Patients. Nephron 2002; 92: 77-85).The present inventors have also revealed that L-PGDS is produced inatherosclerotic plaque and that L-PGDS levels are increased in the bodyfluids of patients with coronary artery disease (Eguchi Y. et al.,Expression of Lipocalin-Type Prostaglandin D Synthase (β-trace) in HumanHeart and its Accumulation in the Coronary Circulation of AnginaPatients. Proc Natl Acad Sci U.S.A. 1997; 94: 14689-94). As describedabove, the relationship between L-PGDS and renal disease or vascularlesion has been revealed. However, the relationship between L-PGDS andPIH has net yet examined.

Non-patent document 1: Hoffmann A. et al., Glycobiology 1997; 7: 499-506

Non-patent document 2: Hamano K. et al., Nephron 2002; 92: 77-85

Non-patent document 3: Eguchi Y. et al., Proc Natl Acad Sci U.S.A. 1997;94: 14689-94

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method forconveniently and objectively determining the severity of PIH, which hasbeen comprehensively determined by various testing methods. Anotherobject of the present invention is to provide a method for predictingthe onset of PIH by precisely detecting abnormalities that occur beforethe onset of PIH (such abnormalities have been impossible to detect byvarious conventional testing methods for PIH) while imposing less of aburden on a subject. Still another object of the present invention is toprovide a method for evaluating a fetus and placental functions in PIH.

As a result of intensive studies to achieve the above objects, thepresent inventors have discovered that the severity of PIH can bedetermined by measuring L-PGDS levels in body fluids such as blood andurine and using the measured values as indexes. Furthermore, the presentinventors have discovered that early prediction of PIH is possible withthe use of such measured values as indexes. Thus, the present inventorshave completed the research.

Specifically, the present invention relates to a method for determiningthe severity of or predicting PIH, which comprises measuring the levelof L-PGDS in a body fluid sample collected from a subject.

Specifically, the present invention is as follows:

-   [1] a method for detecting pregnancy-induced hypertension, which    comprises measuring the level of human lipocalin-type prostaglandin    D synthase in a body fluid sample collected from a subject;-   [2] the method for detecting pregnancy-induced hypertension    according to [1], which comprises measuring the level of human    lipocalin-type prostaglandin D synthase in a body fluid sample    collected from a subject and comparing the measured value with a    cut-off value that is determined based on measured values of human    lipocalin-type prostaglandin D synthase in body fluid samples    collected from normal pregnant women and/or pregnant women with    pregnancy-induced hypertension;-   [3] a method for determining the severity of pregnancy-induced    hypertension, which comprises measuring the level of human    lipocalin-type prostaglandin D synthase in a body fluid sample    collected from a subject;-   [4] the method for determining the severity of pregnancy-induced    hypertension according to [3], which comprises measuring the level    of human lipocalin-type prostaglandin D synthase in a body fluid    sample collected from a subject and comparing the measured value    with cut-off values that are determined according to the measured    values of human lipocalin-type prostaglandin D synthase in the body    fluid samples collected from pregnant women with various severities    of pregnancy-induced hypertension;-   [5] a method for predicting pregnancy-induced hypertension, which    comprises measuring the level of human lipocalin-type prostaglandin    D synthase in a body fluid sample collected from a subject;-   [6] the method for predicting pregnancy-induced hypertension    according to [5], which comprises measuring the level of human    lipocalin-type prostaglandin D synthase in a body fluid sample    collected from a subject showing no hypertension, proteinuria, or    edema;-   [7] the method for predicting pregnancy-induced hypertension    according to [5] or [6], which comprises measuring the level of    human lipocalin-type prostaglandin D synthase in a body fluid sample    collected from a subject and comparing the measured value with a    cut-off value that is determined from measured values of human    lipocalin-type prostaglandin D synthase in body fluid samples    collected from normal pregnant women and/or pregnant women with    pregnancy-induced hypertension;-   [8] a method for evaluating a fetus and a placental function, which    comprises measuring the level of human lipocalin-type prostaglandin    D synthase in a body fluid sample collected from a patient with    pregnancy-induced hypertension;-   [9] the method for detecting pregnancy-induced hypertension    according to [1] or [2], wherein the level of human lipocalin-type    prostaglandin D synthase in a body fluid sample is measured by an    immunological assay method;-   [10] the method for determining the severity of pregnancy-induced    hypertension according to [3] or [4], wherein the level of human    lipocalin-type prostaglandin D synthase in a body fluid sample is    measured by an immunological assay method;-   [11] the method for predicting pregnancy-induced hypertension    according to any one of [5] to [7], wherein the level of human    lipocalin-type prostaglandin D synthase in a body fluid sample is    measured by an immunological assay method;-   [12] the method for evaluating a fetus and a placental function    according to [8], wherein the level of human lipocalin-type    prostaglandin D synthase in a body fluid sample is measured by an    immunological assay method;-   [13] the method according to any one of [1] to [12], wherein the    body fluid sample is blood;-   [14] the method according to any one of [1] to [12], wherein the    body fluid sample is urine; and-   [15] a kit for detecting pregnancy-induced hypertension, which    contains an anti-human lipocalin-type prostaglandin D synthase    antibody.

The present invention will be explained in detail as follows.

This description includes part or all of the contents as disclosed inthe description and/or drawings of Japanese Patent Application No.2003-332084, which is a priority document of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows blood L-PGDS levels in normal pregnant women and pregnantwomen with pregnancy-induced hypertension (PIH) at and before pregnancyweek 31 and at and after pregnancy week 32. In all groups, blood L-PGDSlevels were significantly higher in the subjects with PIH than in thenormal subjects.

FIG. 2 shows the urinary L-PGDS excretions (levels) in normal pregnantwomen and pregnant women with PIH at and before pregnancy week 31 and atand after pregnancy week 32. Spot urine was used as specimen. Eachmeasured L-PGDS value was divided by the urinary creatinine level(L-PGDS per gram of creatinine). In all groups, urinary L-PGDSexcretions were significantly higher in the subjects with PIH than inthe normal subjects.

FIG. 3 shows the results of comparing blood L-PGDS levels in thepregnant women with mild-type and severe-type PIH at pregnancy weeks 26to 38. Severity of PIH was determined by clinical symptoms and the like.Blood L-PGDS levels were significantly higher in the case of severe-typePIH than those in the case of mild-type PIH.

FIG. 4 shows the results of comparing urinary L-PGDS excretions in thepregnant women with mild-type and severe-type PIH at pregnancy weeks 26to 38. Severity of PIH was determined by clinical symptoms and the like.Urinary L-PGDS excretions were significantly higher in the case ofsevere-type PIH than those in the case of mild-type PIH.

FIG. 5 shows the results of measuring L-PGDS levels in conserved seracollected from pregnant women before the onset of PIH. The subjectpregnant women were determined to have mild-type or severe-type PIHbased on clinical symptoms and the like. L-PGDS levels weresignificantly higher in the case of severe-type PIH than those in thecase of mild-type PIH.

FIG. 6 shows the results of measuring urinary L-PGDS excretions inconserved urine collected from pregnant women before the onset of PIH.The subject pregnant women were determined to have mild-type orsevere-type PIH based on clinical symptoms and the like. Spot urine wasused as specimen. Each measured L-PGDS level was divided by the urinarycreatinine concentration (L-PGDS per gram of creatinine). Urinary L-PGDSexcretions were significantly higher in the case of severe-type PIH thanthose in the case of mild-type PIH.

BEST MODE OF CARRYING OUT THE INVENTION

In the present invention, a sample containing L-PGDS to be measured is abody fluid collected from a subject. Specific examples of such sampleinclude blood (e.g., serum or plasma), urine (e.g., spot urine specimensor timed urine specimens), amniotic fluids, cervical mucus, uterineluminal fluids, and oviduct luminal fluids. Of these examples, blood andurine are particularly preferable because they can be easily collected.In this case, a subject may be any pregnant woman regardless of herpregnancy stage (weeks that have passed after conception). Regarding apregnant woman in the early stages of pregnancy, when PIH has not yetbeen developed, her future risk of developing PIH can be determined bythe method of the present invention. Regarding a pregnant woman who hasalready shown symptoms of PIH, the severity of PIH can be determined bythe method of the present invention. A method for measuring L-PGDSlevels in the above samples is not particularly limited, as long as itcan precisely reflect such L-PGDS levels. Examples of such methodinclude an immunological assay method, an enzyme activity assay method,and a capillary electrophoresis method. However, in view of thenecessity of simultaneously and conveniently measuring large amounts ofsamples at an actual clinical site, an immunological assay method usinga monoclonal antibody or a polyclonal antibody specific to L-PGDS, suchas an enzyme immunoassay method, a radio-immunoassay method, a latexagglutination assay method, or a fluorescence immunoassay method ispreferable. A monoclonal antibody that can be preferably used herein isproduced by a hybridoma strain 1B7 (FERM BP-5709), 7F5 (FERM BP-5711),6F5 (FERM BP-5710), 9A6 (FERM BP-5712), 10A3 (FERM BP-5713), or thelike. The hybridoma strain 1B7 was deposited on Sep. 21, 1995 (originaldeposition date) under FERM BP-5709, 7F5 was deposited on Jun. 6, 1996(original deposition date) under FERM BP-5711, 6F5 was deposited on Sep.21, 1995 (original deposition date) under FERM BP-5710, 9A6 wasdeposited on Jun. 6, 1996 (original deposition date) under FERM BP-5712,and 10A3 was deposited on Jun. 6, 1996 (original deposition date) underFERM BP-5713 with the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan). For example, an L-PGDSdetection kit (WO97/16461) that the present inventors have alreadyestablished as a kit to be used for a sandwich ELISA method using amonoclonal antibody may be used.

According to the present invention, PIH at the initial stage can bedetected using an L-PGDS level measured by the above methods as anindicator. Furthermore, severe PIH can be predicted at an early stage.Furthermore, decreased placental functions in the case of PIH can bedetermined using an L-PGDS level measured by the above methods as anindicator.

PIH that is detected by the method of the present invention is limitedto neither preeclampsia, gestational hypertension nor superimposedpreeclampsia. Eclampsia associated with cerebral vasospasm seizure isalso included. Moreover, PIH associated with HELLP (hemolysis, elevatedliver enzymes, and low platelet count) syndrome, which induces pulmonaryedema, encephalorrhagy, premature ablation of normally implantedplacenta, or hepatic vasospasm is also included.

To determine by the method of the present invention whether or not apregnant woman is affected with PIH, first a cut-off value isdetermined. For example, the levels of L-PGDS in body fluid samplescollected from normal pregnant women and/or pregnant women who arealready showing hypertension, proteinuria, or edema as a symptom andhave been clinically diagnosed to have PIH are measured. Then, based ondistribution of L-PGDS level in normal pregnant women or diagnosticaccuracy, such as sensitivity and specificity in the detection of PIH,an appropriate cut-off value for L-PGDS is determined. Subsequently, thelevel of L-PGDS in a body fluid sample collected from a pregnant womanwho is a subject is measured, followed by comparison of the measuredvalue with the cut-off value. When the L-PGDS level in a subject ishigher than the cut-off value, it can be determined that the subject isaffected with PIH. Cut-off values may be previously determined accordingto various severities of PIH. Through comparison of a measured L-PGDSvalue in a subject with each of such cut-off values, the severity of PIHcan be determined. For example, subjects with PIH are divided into agroup of those with severe-type or a group of those with mild-type PIHbased on clinical symptoms such as hypertension, proteinuria, and edema.Distribution of L-PGDS level in each group and diagnostic accuracy fordetermination of the severity are examined, so that appropriate cut-offvalues may be determined. The number of subjects for determination ofthe above cut-off values is not limited. The number of cases ispreferably 5 or more and further preferably 10 or more.

According to the method of the present invention, PIH at the initialstage can also be determined, which is impossible to precisely determinebased only on symptoms such as hypertension, proteinuria, and edema.

A cut-off value to be used in such case is not limited. Specifically, inthe case of blood level, a cut-off value for determining whether or nota subject is affected with PIH can be determined to be between 50 and 70μg/dL, for example, for a pregnant woman at and before pregnancy week 31and can be determined to be between 50 and 60 μg/dL, for example, for apregnant woman at and after pregnancy week 32. Moreover, in the case ofurinary excretion (level), a cut-off value can be determined to bebetween 2.7 and 9 mg/g creatinine for a pregnant woman at and beforepregnancy week 31, and can be determined to be between 3.5 and 7.5 mg/gcreatinine for a pregnant woman at and after pregnancy week 32.Furthermore, a cut-off value for determining if PIH is mild-type orsevere-type is also not limited. For example, in the case of bloodlevel, such a cut-off value can be determined to be between 55 and 70μg/dL. In the case of urinary excretion, such a cut-off value can bedetermined to be between 4 and 9 mg/g creatinine. When a measured L-PGDSvalue in a subject is higher than a cut-off value used for determiningwhether or not a subject is affected with PIH and is lower than acut-off value used for determining if PIH is mild-type or severe-type,it is determined that the relevant subject is affected with mild-typePIH. When a measured value is higher than a cut-off value used fordetermining if PIH is mild-type or severe-type, it is determined thatthe relevant subject is affected with severe-type PIH.

Moreover, the method of the present invention also encompasses a methodfor determining a risk of developing PIH in the pregnant woman who showsnone of hypertension, proteinuria, and edema as symptoms and thus isthought not to be clinically affected with PIH, that is, a method forpredicting PIH. In this case, L-PGDS levels in body fluid samplescollected from normal pregnant women are measured. Subsequently, theconditions of pregnancy of the normal pregnant women are prospectivelyobserved. Such subject pregnant women are divided into a group ofpregnant women who have delivered their babies without developing PIHand a group of pregnant women who have developed PIH during pregnancy. Acut-off value for prediction is determined to be between the measuredL-PGDS values of the former group and the measured L-PGDS values of thelatter group. Alternatively, body fluid samples are collected frompregnant women with PIH before the onset of PIH. Body fluid samples arealso collected from pregnant women who have delivered their babieswithout developing PIH. All of these samples are collected during thesimilar pregnancy period. These samples are stored by means such asfreezing. Through measurement of L-PGDS levels in such stored samples, acut-off value can also be determined retrospectively. Moreover, pregnantwomen are divided by the onset timing of PIH. L-PGDS levels are measuredfor each group of pregnant women and then a cut-off value can bedetermined for each onset timing. Thus, the onset timing of PIH can alsobe predicted. For example, pregnant women are divided into a group ofpregnant women who have developed PIH in the early stage of pregnancy(pregnancy weeks 15 to 25) and a group of pregnant women who havedeveloped PIH in the late stage of pregnancy (at and after pregnancyweek 26). Through measurement of L-PGDS levels in body fluid samples ofeach group, it becomes possible to determine the risk of developing PIHin the early stage of pregnancy and the risk of developing PIH in thelate stage of pregnancy. Furthermore, pregnant women are divided into agroup of pregnant women who have developed mild-type PIH and a group ofpregnant women who have developed severe-type PIH. L-PGDS levels in thebody fluid samples of each group are measured. A cut-off value is thendetermined between L-PGDS levels (measured before the onset) of pregnantwomen who have developed severe-type PIH and L-PGDS levels (measuredbefore onset) of pregnant women who have developed mild-type PIH. Hence,the risk of developing severe-type PIH can be determined.

Subsequently, a body fluid sample is collected from a pregnant womanshowing none of hypertension, proteinuria, and edema as symptoms andthen the L-PGDS level is measured. The measured L-PGDS value is comparedwith the above cut-off value for prediction. Thus, the risk ofdeveloping PIH is determined. For example, when a measured value ishigher than such a cut-off value, it is determined that the risk ofdeveloping PIH in the future is high. When the same is lower than such acut-off value, it is determined that the risk of developing PIH in thefuture is low. Furthermore, when there is no difference between ameasured value and such a cut-off value, determination is suspended.Reexamination may be performed if necessary.

A cut-off value in this case is not limited. For example, a cut-offvalue for predicting whether or not a subject develops PIH duringpregnancy can be determined to be between 55 and 75 μg/dL in the case ofblood level, and between 3.0 and 10 mg/g creatinine in the case ofurinary excretion. Moreover, a cut-off value for predicting whether ornot a subject develops severe-type PIH during pregnancy can bedetermined to be between 60 and 75 μg/dL in the case of blood level andbetween 5.0 and 10 mg/g creatinine in the case of urinary excretion.

As described above, the risk of developing PIH is determined. When suchrisk is determined to be high, the relevant pregnant woman isappropriately treated, so that the risk of developing PIH later or therisk of developing more severe PIH can be reduced. The number ofsubjects needed for determination of the above cut-off value is notlimited. The number of cases is preferably 5 or more and furtherpreferably 10 or more.

Furthermore, the present invention encompasses a method for evaluating afetus and placental functions, which comprises measuring the level ofL-PGDS in a body fluid sample collected from a pregnant woman who hasdeveloped PIH. Here, the term “evaluation of a fetus and placentalfunctions” means evaluation of whether or not placental functions forsupplying nutrition and oxygen to a fetus decrease or means to evaluatewhether or not any injury such as organ damage occurs in the fetus. Whenthe L-PGDS level in a body fluid sample is low, the fetus and placentalfunctions are evaluated to be in a good state. When the same is high,the fetus and placental functions are evaluated to be in a sub-optimalstate.

Furthermore, the present invention encompasses a reagent for detectingor a kit for detecting PIH, which comprises an anti-L-PGDS antibody. Thereagent or the kit may contain a carrier to which an antibody isimmobilized when such kit is based on an enzyme immunoassay method. Suchan antibody may be previously bound to a carrier. Furthermore, the kitmay also appropriately contain a blocking solution, a reaction solution,a stop solution, a reagent for treating a sample, instructionscontaining each cut-off value listed therein, a standard reagent whereinthe L-PGDS level is prepared to be the same as a cut-off value, and thelike.

The present invention will be described in detail by examples asfollows, but the scope of the present invention is not limited by theseexamples.

REFERENCE EXAMPLE Method for Measuring L-PGDS Level in Body Fluid

The L-PGDS level in a body fluid was measured by the sandwich ELISAmethod as follows.

First, an anti-human L-PGDS monoclonal antibody (clone: 7F5) capable ofbinding to human L-PGDS was diluted with a 50 mM carbonate buffer (pH9.6) to a level of 4.4 μg/mL. The solution was added at 300 μL/well to a96-well microtiter plate and then incubation was performed at 4° C.overnight for immobilization. The plate was washed 3 times withphosphate buffered saline (pH 7.4; hereinafter, PBS). Blocking wasperformed by adding PBS containing 0.2% casein (pH 7.4; hereinafter, ablocking solution) at 300 μL/well and then performing incubation at 30°C. for 90 minutes. Subsequently, the plate obtained after blocking waswashed 3 times with PBS containing 0.05% Tween20 (T-PBS). An antigensolution (a standard solution or a body fluid specimen diluted with ablocking solution) was added to the plate at 100 μL/well, followed byincubation at 30° C. for 90 minutes. After reaction, the plate waswashed 3 times with T-PBS. A horseradish-peroxidase-labeled anti-humanL-PGDS monoclonal antibody (clone: 1B7) was diluted with a blockingsolution to a concentration of 0.5 μg/mL. The diluted solution was thenadded at 100 μL/well to the plate and then incubation was performed at30° C. for 90 minutes. After reaction, the plate was washed 3 times withT-PBS. A coloring solution (ABTS solution: produced by BoehringerMannheim Corporation) was added at 100 μL/well, followed by incubationat 30° C. for 30 minutes. After reaction, a stop solution (1.5% oxalicacid) was added at 100 μL/well and then each solution was stirred with aplate mixer, so as to stop the reaction. Absorbance was determined at405 nm using a commercial plate reader.

The monoclonal antibodies (clones: 1B7 and 7F5) used in the abovesandwich ELISA method were prepared as follows. 1.0 mL of pristane wasinjected intraperitoneally into each mouse. 2 weeks after injection,1×10⁸ hybridoma cells producing the 1B7 or 7F5 antibody weretransplanted intraperitoneally into each mouse. An ascites fluid wascollected 2 weeks later and then the obtained ascites fluid wassubjected to protein A affinity column chromatography operation. Inaddition, each cell line producing the above monoclonal antibody agreeswith each monoclonal antibody name. Furthermore, the 1B7 cell line andthe 7F5 cell line were deposited with the International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan) underFERM BP-5709 (original deposition date: Sep. 21, 1995) and under FERMBP-5711 (original deposition date: Jun. 6, 1996), respectively.

EXAMPLE 1

Blood L-PGDS levels of normal pregnant women and pregnant women with PIHwere measured. Subjects were divided into subjects at and beforepregnancy week 31 and subjects at and after pregnancy week 32 dependingon pregnancy stages. The blood L-PGDS levels of the normal pregnantwomen were compared with those of the pregnant women with PIH at eachstage. FIG. 1 shows the results. Regarding the subjects at and beforepregnancy week 31, the L-PGDS level was significantly higher (P<0.0001)in the PIH group (n=10, 72.8±3.8 μg/dL, mean value±standard error) thanthat in the normal group (n=14, 46.1 ±2.8 μg/dL, mean value±standarderror). Moreover, regarding the subjects at and after pregnancy week 32,the L-PGDS level was also significantly higher (P<0.05) in the PIH group(n=10, 65.4±3.1 μg/dL, mean value±standard error) than that in thenormal group (n=13, 48.3±3.8 μg/dL, mean value±standard error). Hence,it was concluded that at any pregnancy stage, a subject showing a highblood L-PGDS level is likely to develop PIH and that measurement ofblood L-PGDS level is useful for detection of PIH.

EXAMPLE 2

The levels of L-PGDS excreted into an urine of normal pregnant women andpregnant women with PIH were measured. Subjects were divided intosubjects at and before pregnancy week 31 and subjects at and afterpregnancy week 32 depending on pregnancy stages. Spot urine specimensfrom the normal pregnant women and the pregnant women with PIH at eachstage were collected. To correct for an influence of the urine volume,each urinary L-PGDS value was divided by urinary creatinine (L-PGDS/gcreatinine). FIG. 2 shows the results. Regarding the subjects at andbefore pregnancy week 31, the L-PGDS excretion was significantly higher(P<0.0001) in the PIH group (n=10, 9.90±2.24 mg/g creatinine, meanvalue±standard error) than that in the normal group (n=14, 2.53±0.26mg/g creatinine, mean value±standard error). Moreover, regarding thesubjects at and after pregnancy week 32, the L-PGDS excretion was alsosignificantly higher (P<0.005) in the PIH group (n=10, 8.03±1.11 mg/gcreatinine, mean value±standard error) than that in the normal group(n=14, 3.38±0.58 mg/g creatinine, mean value±standard error). Hence, itwas concluded that at any pregnancy stage, a subject showing a highurinary L-PGDS excretion is likely to develop PIH and that measurementof urinary L-PGDS excretion is useful for detection of PIH.

EXAMPLE 3

Blood L-PGDS levels of pregnant women with PIH at pregnancy weeks 26 to38 were measured. Subjects were divided into subjects with mild-type PIHand subjects with severe-type PIH based on hypertension, proteinuria,edema, other clinical symptoms, and the like. The blood L-PGDS levelsthereof were compared. As a result, as shown in FIG. 3, the blood L-PGDSlevel was significantly higher (P<0.01) in severe-type group (n=12,71.9±3.6 μg/dL, mean value±standard error) than that in mild-type group(n=9, 52.4±4.8 μg/dL, mean value±standard error). Hence, it wasconcluded that PIH is likely to be severe when a patient with PIH showsa high blood L-PGDS level and that measurement of blood L-PGDS level isuseful in determination of the severity of PIH.

EXAMPLE 4

The levels of L-PGDS excreted into an urine of pregnant women with PIHat pregnancy weeks 26 to 38 were measured. Subjects were divided intosubjects with mild-type PIH and subjects with severe-type PIH based onhypertension, proteinuria, edema, other clinical symptoms, and the like.The urinary L-PGDS excretions thereof were compared. Spot urine was usedas specimen. Each urinary L-PGDS value was divided by the urinarycreatinine concentration (L-PGDS per gram of creatinine). As a result,as shown in FIG. 4, the urinary L-PGDS excretion was significantlyhigher (P<0.01) in severe-type group (n=12, 9.72±3.46 mg/g creatinine,mean value±standard error) than that in mild-type group (n=9, 3.87±1.18mg/g creatinine, mean value±standard error). Hence, it was concludedthat PIH is likely to be severe when a patient with PIH shows a highurinary L-PGDS excretion and that measurement of urinary L-PGDSexcretion is useful in determination of the severity of PIH.

EXAMPLE 5

Blood L-PGDS levels of 24 pregnant women at pregnancy weeks 15 to 25,who had not developed PIH, were measured. During the period from theL-PGDS measurement to delivery, the subjects were prospectively observedto determine the presence or the absence of the onset of PIH. Thepresence or the absence of the onset of PIH was determinedcomprehensively based on hypertension, proteinuria, edema, otherclinical symptoms, and the like. A provisional cut-off value was set at61.8 μg/dL, which was the 95 percentile of blood L-PGDS levels in normalpregnant women at and before pregnancy week 31 as shown in Example 1.The subjects were classified into a group of subjects showing L-PGDSlevels equal to or lower than the cut-off value and a group of subjectsshowing L-PGDS levels higher than the cut-off value. As a result, asshown in Table 1, whereas only 1 out of 17 subjects developed PIH (5.9%)in the group of subjects showing blood L-PGDS levels equal to or lowerthan 61.8 μg/dL, 2 out of 7 subjects developed PIH (as high as 28.6%) inthe group of subjects showing L-PGDS levels higher than 61.8 μg/dL.Based on this result, it was concluded that even in the case of apregnant woman who has not developed PIH, such a subject is likely todevelop PIH when her blood L-PGDS level is high. It was also concludedthat the onset of PIH can be predicted by measuring blood L-PGDS level.TABLE 1 Blood L-PGDS level and the following onset of PIH Case No. BloodL-PGDS (μg/dL) Onset of PIH 1 40 No 2 64.6 Yes 3 51.1 No 4 38.6 No 539.0 No 6 37.8 No 7 59.5 Yes 8 58.8 No 9 49.5 No 10 76.0 No 11 39.8 No12 53.5 No 13 64.8 No 14 46.8 No 15 50.5 No 16 68.2 No 17 43.0 No 1848.7 No 19 34.6 No 20 62.3 No 21 44.8 No 22 59.6 No 23 64.3 No 24 78.8Yes L-PGDS > 61.8 μg/dL

EXAMPLE 6

The levels of L-PGDS excreted into an urine of 35 pregnant women atweeks 15 to 25, who had not developed PIH, were measured. During theperiod from the L-PGDS measurement to delivery, the subjects wereprospectively observed to determine the presence or the absence of theonset of PIH. The presence or the absence of the of the onset of PIH wasdetermined comprehensively based on hypertension, proteinuria, edema,other clinical symptoms, and the like. Spot urine was used as specimen.Each urinary L-PGDS value was divided by the urinary creatinineconcentration (L-PGDS per gram of creatinine). A provisional cut-offvalue was set at 4.21 mg/g creatinine, which was the 95 percentile ofurinary L-PGDS excretions in normal pregnant women at and beforepregnancy week 31 as shown in Example 2. The subjects were classifiedinto a group of subjects showing L-PGDS excretions equal to or lowerthan the cut-off value and a group of subjects showing L-PGDS excretionshigher than the cut-off value. As a result, as shown in Table 2, whereasonly 2 out of 26 subjects developed PIH (7.7%) in the group of subjectsshowing urinary L-PGDS excretions equal to or lower than 4.21 mg/gcreatinine, 3 out of 9 subjects developed PIH (as high as 33.3%) in thegroup of subjects showing L-PGDS excretions higher than 4.21 mg/gcreatinine. Based on this result, it was concluded that even in the caseof a pregnant woman who has not developed PIH, such a subject is likelyto develop PIH when her urinary L-PGDS excretion is high. It was alsoconcluded that the onset of PIH can be predicted by measuring urinaryL-PGDS excretion. TABLE 2 Urinary L-PGDS excretion and the followingonset of PIH Urinary L-PGDS Case No. (mg/g Creatinine) Onset of PIH 10.96 No 2 1.45 No 3 11.21 Yes 4 6.63 No 5 3.72 No 6 4.51 No 7 0.97 No 82.40 No 9 1.46 No 10 2.91 Yes 11 1.90 No 12 1.12 No 13 0.27 No 14 1.01No 15 1.05 No 16 4.41 No 17 3.89 No 18 4.50 Yes 19 1.61 No 20 1.61 No 211.57 No 22 2.94 No 23 2.26 No 24 2.06 No 25 3.72 No 26 0.13 No 27 0.80No 28 4.75 No 29 1.80 No 30 4.17 Yes 31 4.78 No 32 6.69 No 33 3.49 No 342.16 No 35 12.98 Yes L-PGDS > 4.21 mg/g Creatinine

EXAMPLE 7

L-PGDS levels in conserved sera collected (during pregnancy weeks 15 to25) from 17 pregnant women who had developed PIH at and after pregnancyweek 26 were measured. As a result, as shown in FIG. 5, it was revealedthat the L-PGDS level was significantly higher (P<0.05) in the case ofsevere-type PIH (n=8, 69.2±2.6 μg/dL, mean value±standard error) thanthat in the case of mild-type PIH (n=9, 58.9±3.2 μg/dL, meanvalue±standard error). Hence, it was concluded that a patient showing ahigh blood L-PGDS level is likely to develop severe-type PIH and thatmeasurement of blood L-PGDS level is useful in prediction of severe-typePIH.

EXAMPLE 8

L-PGDS excretions in conserved spot urine collected (during pregnancyweeks 15 to 25) from 17 pregnant women who had developed PIH at andafter pregnancy week 26 were measured. As a result, as shown in FIG. 6,it was revealed that the L-PGDS excretion was significantly higher(P<0.05) in the case of severe-type PIH (n=8, 8.69±0.96 mg/g creatinine,mean value±standard error) than that in the case of mild-type PIH (n=8,4.59±0.75 mg/g creatinine, mean value±standard error). Hence, it wasconcluded that a patient showing a high urinary L-PGDS excretion islikely to develop severe-type PIH and that measurement of urinary L-PGDSexcretion is useful in prediction of severe-type PIH.

INDUSTRIAL APPLICABILITY

According to the present invention, a method by which PIH can bedetected conveniently while imposing less of a burden on a subject isprovided. Furthermore, by the use of the method of the presentinvention, the severity of PIH (that has been determined comprehensivelyusing various testing methods) can be conveniently and objectivelydetermined. Furthermore, by the use of the method of the presentinvention, the risk of developing PIH in a pregnant woman who has notshown clinical symptoms such as hypertension, proteinuria, or edema canalso be predicted. Therefore, the method of the present invention isextremely useful for determining the severity of and predicting PIH.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. A method for detecting pregnancy-induced hypertension, whichcomprises measuring the level of human lipocalin-type prostaglandin Dsynthase in a body fluid sample collected from a subject.
 2. The methodfor detecting pregnancy-induced hypertension according to claim 1, whichcomprises measuring the level of human lipocalin-type prostaglandin Dsynthase in a body fluid sample collected from a subject and comparingthe measured value with a cut-off value that is determined based onmeasured values of human lipocalin-type prostaglandin D synthase in bodyfluid samples collected from normal pregnant women and/or pregnant womenwith pregnancy-induced hypertension.
 3. A method for determining theseverity of pregnancy-induced hypertension, which comprises measuringthe level of human lipocalin-type prostaglandin D synthase in a bodyfluid sample collected from a subject.
 4. The method for determining theseverity of pregnancy-induced hypertension according to claim 3, whichcomprises measuring the level of human lipocalin-type prostaglandin Dsynthase in a body fluid sample collected from a subject and comparingthe measured value with cut-off values that are determined according tothe measured values of human lipocalin-type prostaglandin D synthase inthe body fluid samples collected from pregnant women with variousseverities of pregnancy-induced hypertension.
 5. A method for predictingpregnancy-induced hypertension, which comprises measuring the level ofhuman lipocalin-type prostaglandin D synthase in a body fluid samplecollected from a subject.
 6. The method for predicting pregnancy-inducedhypertension according to claim 5, which comprises measuring the levelof human lipocalin-type prostaglandin D synthase in a body fluid samplecollected from a subject showing no hypertension, proteinuria, or edema.7. The method for predicting pregnancy-induced hypertension according toclaim 5, which comprises measuring the level of human lipocalin-typeprostaglandin D synthase in a body fluid sample collected from a subjectand comparing the measured value with a cut-off value that is determinedfrom measured values of human lipocalin-type prostaglandin D synthase inbody fluid samples collected from normal pregnant women and/or pregnantwomen with pregnancy-induced hypertension.
 8. A method for evaluating afetus and a placental function, which comprises measuring the level ofhuman lipocalin-type prostaglandin D synthase in a body fluid samplecollected from a patient with pregnancy-induced hypertension.
 9. Themethod for detecting pregnancy-induced hypertension according to claim1, wherein the level of human lipocalin-type prostaglandin D synthase ina body fluid sample is measured by an immunological assay method. 10.The method for determining the severity of pregnancy-inducedhypertension according to claim 3, wherein the level of humanlipocalin-type prostaglandin D synthase in a body fluid sample ismeasured by an immunological assay method.
 11. The method for predictingpregnancy-induced hypertension according to claim 5, wherein the levelof human lipocalin-type prostaglandin D synthase in a body fluid sampleis measured by an immunological assay method.
 12. The method forevaluating a fetus and a placental function according to claim 8,wherein the level of human lipocalin-type prostaglandin D synthase in abody fluid sample is measured by an immunological assay method.
 13. Themethod according to claim 1, wherein the body fluid sample is blood. 14.The method according to claim 1, wherein the body fluid sample is urine.15. A kit for detecting pregnancy-induced hypertension, which containsan anti-human lipocalin-type prostaglandin D synthase antibody.